Compositions and methods for identifying and selecting maize plants with resistance to northern leaf blight

ABSTRACT

Compositions and methods useful in identifying and/or selecting maize plants having resistance to northern leaf blight are provided herein. The resistance may be newly conferred or enhanced relative to a control plant. The methods use markers to identify, select and/or construct resistant plants. Maize plants identified, selected, and/or generated by the methods described herein are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/502,696 filed Feb. 8, 2017, which claims the benefit fromInternational Application No. PCT/US2015/043529 filed Aug. 4, 2015,which claims the benefit of U.S. Provisional Application No. 62/034,806,filed Aug. 8, 2014, the entire contents of each are herein incorporatedby reference.

FIELD

The field is related to plant breeding and methods of identifying andselecting plants with resistance to northern leaf blight.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20150715_BB1987PCT_SequenceListing_ST25.txt created on Jul. 15, 2015 andhaving a size of 10 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Northern leaf blight (NLB) caused by the fungus Setosphaeria turcica(also known as Exserohilum turcicum or Helminthosporium turcicum) is amajor disease of maize in North America, South America, Africa and Asia.Symptoms can range from cigar-shaped lesions on the lower leaves tocomplete destruction of the foliage, thereby reducing the amount of leafsurface area available for photosynthesis which in turn impacts grainyield. Disease management strategies include crop rotation, destructionof old maize residues by tillage, and fungicide application, all ofwhich are aimed at reducing the fungal inoculum. However, the mosteffective and most preferred method of control for northern leaf blightis the planting of resistant hybrids.

Several varieties or races of Exserohilum turcicum are present innature, leaving growers with two hybrid options: partial resistanthybrids, which offer low-level, broad spectrum protection againstmultiple races, and race-specific resistant hybrids, which protectagainst a specific race. Genetic sources of Exserohilum turcicum havebeen described, and four Exserohilum turcicum (previously calledHelminthosporium turcicum) resistance loci have been identified: Ht1,Ht2, Ht3, and Htn1. Gene Ht1 maps to the long arm of chromosome 2 whereit is closely linked to umc36 (Coe, E. H. et al. (1988), Corn and CornImprovement, 3rd edn., pp. 81-258), sgcr506 (Gupta, M. et al. (1989)Maize Genet. Coop. Newsl. 63, 112), umc150B (Bentolila, S. et al. (1991)Theor. Appl. Genet., 82:393-398), and pic18a (Collins et al. (1998)Molecular Plant-Microbe Interactions, 11:968-978), and it is closelyflanked by umc22 and umc122 (Li et al. (1998) Hereditas, 129:101-106).Gene Ht2 maps to the long arm of chromosome 8 in the umc48-umc89interval (Zaitlin et al. (1992) Maize Genet. Coop. Newsl., 66, 69-70),and gene Ht3 maps to chromosome 7 near bn1g1666 (Van Staden, D et al.(2001) Maize Genetics Conference Abstracts 43:P134). The Htn1 gene mapsto chromosome 8, approximately 10 cM distal to Ht2 and 0.8 cM distal tothe RFLP marker umc117 (Simcox and Bennetzen (1993) Maize Genet. Coop.Newl. 67, 118-119; Simcox and Bennetzen (1993) Phytopathology,83:1326-1330; Chung et al. (2010) Theor App Gen Epub).

Since the QTL respond to different races and each QTL has a variableeffect on the northern leaf blight resistance trait, it is desirable toidentify new sources of genetic resistance that can be combined withother known resistance loci to enhance overall resistance to northernleaf blight.

SUMMARY

Compositions and methods for identifying and selecting maize plants withenhanced resistance to northern leaf blight are provided.

Methods for identifying maize plants with northern leaf blightresistance are provided herein. The methods involve analyzing DNA of amaize plant for the presence of a QTL allele associated with northernleaf blight resistance and selecting maize plants as having northernleaf blight resistance if the QTL allele is detected. The QTL allele islocated within an interval on chromosome 5 comprising and flanked byPHM18056 and PHM7958 and may comprise: a “G” at PZE-105068275; an “A” atPZE-105068432; a “C” at PZE-105068572; a “T” at SYN30642; a “C” atPZE-105068746; an “A” at PZE-105069095; an “A” at PZE-105069706; a “T”at PZE-105069906; and a “C” at PZE-105070525. A subinterval of theinterval in which the QTL allele is located may be further defined bymarkers PZE-105068275 and PZE-105070525.

Methods for introgressing a QTL allele associated with northern leafblight resistance into a maize plant are provided. The methods involvescreening a population with at least one marker to determine if one ormore maize plants from the population comprises a QTL allele associatedwith northern leaf blight resistance and selecting from the populationone or more maize plants that have the QTL allele. The QTL allele maycomprise: a “G” at PZE-105068275; an “A” at PZE-105068432; a “C” atPZE-105068572; a “T” at SYN30642; a “C” at PZE-105068746; an “A” atPZE-105069095; an “A” at PZE-105069706; a “T” at PZE-105069906; and a“C” at PZE-105070525.

Plants identified, selected, or produced by the methods described hereinare also provided.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. .sctn.1.822.

FIG. 1 shows the diagram used as a guide to score northern leaf blightinfection.

SEQ ID NO:1 is the reference sequence for marker PHM16750.

SEQ ID NO:2 is the reference sequence for marker PHM15741.

SEQ ID NO:3 is the reference sequence for marker PHM16854.

SEQ ID NO:4 is the reference sequence for marker PHM3870.

SEQ ID NO:5 is the reference sequence for marker PHM14018.

SEQ ID NO:6 is the reference sequence for marker PHM18056.

SEQ ID NO:7 is the reference sequence for marker PHM3467.

SEQ ID NO:8 is the reference sequence for marker PHM7958.

SEQ ID NO:9 is the reference sequence for marker PZE-105068275.

SEQ ID NO:10 is the reference sequence for marker PZE-105068432.

SEQ ID NO:11 is the reference sequence for marker PZE-105068572.

SEQ ID NO:12 is the reference sequence for marker SYN30642.

SEQ ID NO:13 is the reference sequence for marker PZE-105068746.

SEQ ID NO:14 is the reference sequence for marker PZE-105069095.

SEQ ID NO:15 is the reference sequence for marker PZE-105069706.

SEQ ID NO:16 is the reference sequence for marker PZE-105069906.

SEQ ID NO:17 is the reference sequence for marker PZE-105070525.

DETAILED DESCRIPTION

Maize marker loci that demonstrate statistically significantco-segregation with the northern leaf blight resistance trait areprovided herein. Detection of these loci or additional linked loci canbe used in marker assisted selection as part of a maize breeding programto produce maize plants that have resistance to northern leaf blight,which is caused by the pathogen Exserohilum turcicum.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation. Numeric ranges recited within the specificationare inclusive of the numbers defining the range and include each integeror any non-integer fraction within the defined range. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the disclosure pertains. Methods and materials similar orequivalent to those described herein can be used in the practice fortesting of the subject matter presented herein. In describing andclaiming the present invention, the following terminology will be usedin accordance with the definitions set out below.

The following definitions are provided as an aid to understand thepresent disclosure.

It is to be understood that the disclosure is not limited to particularembodiments, which can, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting. As usedin this specification and the appended claims, terms in the singular andthe singular forms “a”, “an” and “the”, for example, include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “plant”, “the plant” or “a plant” also includes aplurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny ofthat plant; use of the term “a nucleic acid” optionally includes, as apractical matter, many copies of that nucleic acid molecule; similarly,the term “probe” optionally (and typically) encompasses many similar oridentical probe molecules.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus.

“Allele frequency” refers to the frequency (proportion or percentage) atwhich an allele is present at a locus within an individual, within aline, or within a population of lines. For example, for an allele “A”,diploid individuals of genotype “AA”, “Aa”, or “aa” have allelefrequencies of 1.0, 0.5, or 0.0, respectively. One can estimate theallele frequency within a line by averaging the allele frequencies of asample of individuals from that line. Similarly, one can calculate theallele frequency within a population of lines by averaging the allelefrequencies of lines that make up the population. For a population witha finite number of individuals or lines, an allele frequency can beexpressed as a count of individuals or lines (or any other specifiedgrouping) containing the allele.

An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that isproduced by amplifying a template nucleic acid by any availableamplification method (e.g., PCR, LCR, transcription, or the like).

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods.

The term “assemble” applies to BACs and their propensities for comingtogether to form contiguous stretches of DNA. A BAC “assembles” to acontig based on sequence alignment, if the BAC is sequenced, or via thealignment of its BAC fingerprint to the fingerprints of other BACs.Public assemblies can be found using the Maize Genome Browser, which ispublicly available on the internet.

An allele is “associated with” a trait when it is part of or linked to aDNA sequence or allele that affects the expression of the trait. Thepresence of the allele is an indicator of how the trait will beexpressed.

A “BAC”, or bacterial artificial chromosome, is a cloning vector derivedfrom the naturally occurring F factor of Escherichia coli, which itselfis a DNA element that can exist as a circular plasmid or can beintegrated into the bacterial chromosome. BACs can accept large insertsof DNA sequence. In maize, a number of BACs each containing a largeinsert of maize genomic DNA from maize inbred line B73, have beenassembled into contigs (overlapping contiguous genetic fragments, or“contiguous DNA”), and this assembly is available publicly on theinternet.

A BAC fingerprint is a means of analyzing similarity between several DNAsamples based upon the presence or absence of specific restriction sites(restriction sites being nucleotide sequences recognized by enzymes thatcut or “restrict” the DNA). Two or more BAC samples are digested withthe same set of restriction enzymes and the sizes of the fragmentsformed are compared, usually using gel separation.

“Backcrossing” refers to the process whereby hybrid progeny arerepeatedly crossed back to one of the parents. In a backcrossing scheme,the “donor” parent refers to the parental plant with the desiredgene/genes, locus/loci, or specific phenotype to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. For example, see Ragot, M. et al. (1995)Marker-assisted backcrossing: a practical example, in Techniques etUtilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp.45-56, and Openshaw et al., (1994) Marker-assisted Selection inBackcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. Theinitial cross gives rise to the F1 generation; the term “BC1” thenrefers to the second use of the recurrent parent, “BC2” refers to thethird use of the recurrent parent, and so on.

A centimorgan (“cM”) is a unit of measure of recombination frequency.One cM is equal to a 1% chance that a marker at one genetic locus willbe separated from a marker at a second locus due to crossing over in asingle generation.

As used herein, the term “chromosomal interval” designates a contiguouslinear span of genomic DNA that resides in planta on a singlechromosome. The genetic elements or genes located on a singlechromosomal interval are physically linked. The size of a chromosomalinterval is not particularly limited. In some aspects, the geneticelements located within a single chromosomal interval are geneticallylinked, typically with a genetic recombination distance of, for example,less than or equal to 20 cM, or alternatively, less than or equal to 10cM. That is, two genetic elements within a single chromosomal intervalundergo recombination at a frequency of less than or equal to 20% or10%.

A “chromosome” is a single piece of coiled DNA containing many genesthat act and move as a unity during cell division and therefore can besaid to be linked. It can also be referred to as a “linkage group”.

The phrase “closely linked”, in the present application, means thatrecombination between two linked loci occurs with a frequency of equalto or less than about 10% (i.e., are separated on a genetic map by notmore than 10 cM). Put another way, the closely linked loci co-segregateat least 90% of the time. Marker loci are especially useful with respectto the subject matter of the current disclosure when they demonstrate asignificant probability of co-segregation (linkage) with a desired trait(e.g., resistance to northern leaf blight). Closely linked loci such asa marker locus and a second locus can display an inter-locusrecombination frequency of 10% or less, preferably about 9% or less,still more preferably about 8% or less, yet more preferably about 7% orless, still more preferably about 6% or less, yet more preferably about5% or less, still more preferably about 4% or less, yet more preferablyabout 3% or less, and still more preferably about 2% or less. In highlypreferred embodiments, the relevant loci display a recombination afrequency of about 1% or less, e.g., about 0.75% or less, morepreferably about 0.5% or less, or yet more preferably about 0.25% orless. Two loci that are localized to the same chromosome, and at such adistance that recombination between the two loci occurs at a frequencyof less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1° A,0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” eachother. In some cases, two different markers can have the same geneticmap coordinates. In that case, the two markers are in such closeproximity to each other that recombination occurs between them with suchlow frequency that it is undetectable.

The term “complement” refers to a nucleotide sequence that iscomplementary to a given nucleotide sequence, i.e. the sequences arerelated by the Watson-Crick base-pairing rules.

The term “contiguous DNA” refers to an uninterrupted stretch of genomicDNA represented by partially overlapping pieces or contigs.

When referring to the relationship between two genetic elements, such asa genetic element contributing to northern leaf blight resistance and aproximal marker, “coupling” phase linkage indicates the state where the“favorable” allele at the northern leaf blight resistance locus isphysically associated on the same chromosome strand as the “favorable”allele of the respective linked marker locus. In coupling phase, bothfavorable alleles are inherited together by progeny that inherit thatchromosome strand.

The term “crossed” or “cross” refers to a sexual cross and involved thefusion of two haploid gametes via pollination to produce diploid progeny(e.g., cells, seeds or plants). The term encompasses both thepollination of one plant by another and selfing (or self-pollination,e.g., when the pollen and ovule are from the same plant).

A plant referred to herein as “diploid” has two sets (genomes) ofchromosomes.

A plant referred to herein as a “doubled haploid” is developed bydoubling the haploid set of chromosomes (i.e., half the normal number ofchromosomes). A doubled haploid plant has two identical sets ofchromosomes, and all loci are considered homozygous.

An “elite line” is any line that has resulted from breeding andselection for superior agronomic performance.

An “exotic maize strain” or an “exotic maize germ plasm” is a strainderived from a maize plant not belonging to an available elite maizeline or strain of germplasm. In the context of a cross between two maizeplants or strains of germplasm, an exotic germ plasm is not closelyrelated by descent to the elite germplasm with which it is crossed. Mostcommonly, the exotic germplasm is not derived from any known elite lineof maize, but rather is selected to introduce novel genetic elements(typically novel alleles) into a breeding program.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, an agronomically desirable phenotype, e.g., northernleaf blight resistance, and that allows the identification of plantswith that agronomically desirable phenotype. A favorable allele of amarker is a marker allele that segregates with the favorable phenotype.

“Fragment” is intended to mean a portion of a nucleotide sequence.Fragments can be used as hybridization probes or PCR primers usingmethods disclosed herein.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form. For eachgenetic map, distances between loci are measured by how frequently theiralleles appear together in a population (their recombinationfrequencies). Alleles can be detected using DNA or protein markers, orobservable phenotypes. A genetic map is a product of the mappingpopulation, types of markers used, and the polymorphic potential of eachmarker between different populations. Genetic distances between loci candiffer from one genetic map to another. However, information can becorrelated from one map to another using common markers. One of ordinaryskill in the art can use common marker positions to identify positionsof markers and other loci of interest on each individual genetic map.The order of loci should not change between maps, although frequentlythere are small changes in marker orders due to e.g. markers detectingalternate duplicate loci in different populations, differences instatistical approaches used to order the markers, novel mutation orlaboratory error.

A “genetic map location” is a location on a genetic map relative tosurrounding genetic markers on the same linkage group where a specifiedmarker can be found within a given species.

“Genetic mapping” is the process of defining the linkage relationshipsof loci through the use of genetic markers, populations segregating forthe markers, and standard genetic principles of recombination frequency.

“Genetic markers” are nucleic acids that are polymorphic in a populationand where the alleles of which can be detected and distinguished by oneor more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and thelike. The term also refers to nucleic acid sequences complementary tothe genomic sequences, such as nucleic acids used as probes. Markerscorresponding to genetic polymorphisms between members of a populationcan be detected by methods well-established in the art. These include,e.g., PCR-based sequence specific amplification methods, detection ofrestriction fragment length polymorphisms (RFLP), detection of isozymemarkers, detection of polynucleotide polymorphisms by allele specifichybridization (ASH), detection of amplified variable sequences of theplant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also know forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and randomly amplified polymorphic DNA (RAPD).

“Genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis.

“Genome” refers to the total DNA, or the entire set of genes, carried bya chromosome or chromosome set.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci. Genotype is definedby the allele(s) of one or more known loci that the individual hasinherited from its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture, or moregenerally, all individuals within a species or for several species(e.g., maize germplasm collection or Andean germplasm collection). Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells, which can be cultured into a whole plant.

A plant referred to as “haploid” has a single set (genome) ofchromosomes.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e. a combination of alleles. Typically, the genetic locidescribed by a haplotype are physically and genetically linked, i.e., onthe same chromosome segment. The term “haplotype” can refer to allelesat a particular locus, or to alleles at multiple loci along achromosomal segment.

The term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

The heterotic response of material, or “heterosis”, can be defined byperformance which exceeds the average of the parents (or high parent)when crossed to other dissimilar or unrelated groups.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group (Hallauer et al.(1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley(ed.) Corn and corn improvement). Inbred lines are classified intoheterotic groups, and are further subdivided into families within aheterotic group, based on several criteria such as pedigree, molecularmarker-based associations, and performance in hybrid combinations (Smithet al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely usedheterotic groups in the United States are referred to as “Iowa StiffStalk Synthetic” (also referred to herein as “stiff stalk”) and“Lancaster or “Lancaster Sure Crop” (sometimes referred to as NSS, ornon-Stiff Stalk).

Some heterotic groups possess the traits needed to be a female parent,and others, traits for a male parent. For example, in maize, yieldresults from public inbreds released from a population called BSSS (IowaStiff Stalk Synthetic population) has resulted in these inbreds andtheir derivatives becoming the female pool in the central Corn Belt.BSSS inbreds have been crossed with other inbreds, e.g. SD 105 and MaizAmargo, and this general group of materials has become known as StiffStalk Synthetics (SSS) even though not all of the inbreds are derivedfrom the original BSSS population (Mikel and Dudley (2006) Crop Sci:46:1193-1205). By default, all other inbreds that combine well with theSSS inbreds have been assigned to the male pool, which for lack of abetter name has been designated as NSS, i.e. Non-Stiff Stalk. This groupincludes several major heterotic groups such as Lancaster Surecrop,lodent, and Leaming Corn.

An individual is “heterozygous” if more than one allele type is presentat a given locus (e.g., a diploid individual with one copy each of twodifferent alleles).

The term “homogeneity” indicates that members of a group have the samegenotype at one or more specific loci.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes).

The term “hybrid” refers to the progeny obtained between the crossing ofat least two genetically dissimilar parents.

“Hybridization” or “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands.

The term “hybridize” means to form base pairs between complementaryregions of nucleic acid strands.

An “IBM genetic map” can refer to any of following maps: IBM, IBM2, IBM2neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2 2005 neighbors, IBM22005 neighbors frame, IBM2 2008 neighbors, IBM2 2008 neighbors frame, orthe latest version on the maizeGDB website. IBM genetic maps are basedon a B73×Mo17 population in which the progeny from the initial crosswere random-mated for multiple generations prior to constructingrecombinant inbred lines for mapping. Newer versions reflect theaddition of genetic and BAC mapped loci as well as enhanced maprefinement due to the incorporation of information obtained from othergenetic maps or physical maps, cleaned date, or the use of newalgorithms.

The term “inbred” refers to a line that has been bred for genetichomogeneity.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an inserted nucleotide or piece of DNArelative to a second line or the second line may be referred to ashaving a deleted nucleotide or piece of DNA relative to the first line.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g.,detected by a marker that is associated with a phenotype, at a QTL (i.e.a QTL allele), a transgene, or the like. In any case, offspringcomprising the desired allele can be repeatedly backcrossed to a linehaving a desired genetic background and selected for the desired allele,to result in the allele becoming fixed in a selected genetic background.

The process of “introgressing” is often referred to as “backcrossing”when the process is repeated two or more times.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is associated with another marker locus or someother locus. The linkage relationship between a molecular marker and alocus affecting a phenotype is given as a “probability” or “adjustedprobability”. Linkage can be expressed as a desired limit or range. Forexample, in some embodiments, any marker is linked (genetically andphysically) to any other marker when the markers are separated by lessthan 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map(a genetic map based on a population that has undergone one round ofmeiosis, such as e.g. an F2; the IBM2 maps consist of multiple meioses).In some aspects, it is advantageous to define a bracketed range oflinkage, for example, between 10 and 20 cM, between 10 and 30 cM, orbetween 10 and 40 cM. The more closely a marker is linked to a secondlocus, the better an indicator for the second locus that marker becomes.Thus, “closely linked loci” such as a marker locus and a second locusdisplay an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be“in proximity to” each other. Since one cM is the distance between twomarkers that show a 1% recombination frequency, any marker is closelylinked (genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” (or LD) refers to a non-randomsegregation of genetic loci or traits (or both). In either case, linkagedisequilibrium implies that the relevant loci are within sufficientphysical proximity along a length of a chromosome so that they segregatetogether with greater than random (i.e., non-random) frequency. Markersthat show linkage disequilibrium are considered linked. Linked locico-segregate more than 50% of the time, e.g., from about 51% to about100% of the time. In other words, two markers that co-segregate have arecombination frequency of less than 50% (and by definition, areseparated by less than 50 cM on the same linkage group.) As used herein,linkage can be between two markers, or alternatively between a markerand a locus affecting a phenotype. A marker locus can be “associatedwith” (linked to) a trait. The degree of linkage of a marker locus and alocus affecting a phenotypic trait is measured, e.g., as a statisticalprobability of co-segregation of that molecular marker with thephenotype (e.g., an F statistic or LOD score).

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor. Appl. Genet. 38:226-231(1968). When r²=1, completeLD exists between the two marker loci, meaning that the markers have notbeen separated by recombination and have the same allele frequency. Ther² value will be dependent on the population used. Values for r² above ⅓indicate sufficiently strong LD to be useful for mapping (Ardlie et al.,Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkagedisequilibrium when r² values between pairwise marker loci are greaterthan or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

A “locus” is a position on a chromosome, e.g. where a nucleotide, gene,sequence, or marker is located.

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science255:803-804 (1992)) is used in genetic interval mapping to describe thedegree of linkage between two marker loci. A LOD score of three betweentwo markers indicates that linkage is 1000 times more likely than nolinkage, while a LOD score of two indicates that linkage is 100 timesmore likely than no linkage. LOD scores greater than or equal to two maybe used to detect linkage. LOD scores can also be used to show thestrength of association between marker loci and quantitative traits in“quantitative trait loci” mapping. In this case, the LOD score's size isdependent on the closeness of the marker locus to the locus affectingthe quantitative trait, as well as the size of the quantitative traiteffect.

“Maize” refers to a plant of the Zea mays L. ssp. mays and is also knownas “corn”.

The term “maize plant” includes whole maize plants, maize plant cells,maize plant protoplast, maize plant cell or maize tissue culture fromwhich maize plants can be regenerated, maize plant calli, maize plantclumps and maize plant cells that are intact in maize plants or parts ofmaize plants, such as maize seeds, maize cobs, maize flowers, maizecotyledons, maize leaves, maize stems, maize buds, maize roots, maizeroot tips and the like.

A “marker” is a means of finding a position on a genetic or physicalmap, or else linkages among markers and trait loci (loci affectingtraits). The position that the marker detects may be known via detectionof polymorphic alleles and their genetic mapping, or else byhybridization, sequence match or amplification of a sequence that hasbeen physically mapped. A marker can be a DNA marker (detects DNApolymorphisms), a protein (detects variation at an encoded polypeptide),or a simply inherited phenotype (such as the ‘waxy’ phenotype). A DNAmarker can be developed from genomic nucleotide sequence or fromexpressed nucleotide sequences (e.g., from a spliced RNA or a cDNA).Depending on the DNA marker technology, the marker will consist ofcomplementary primers flanking the locus and/or complementary probesthat hybridize to polymorphic alleles at the locus. A DNA marker, or agenetic marker, can also be used to describe the gene, DNA sequence ornucleotide on the chromosome itself (rather than the components used todetect the gene or DNA sequence) and is often used when that DNA markeris associated with a particular trait in human genetics (e.g. a markerfor breast cancer). The term marker locus is the locus (gene, sequenceor nucleotide) that the marker detects.

Markers that detect genetic polymorphisms between members of apopulation are well-established in the art. Markers can be defined bythe type of polymorphism that they detect and also the marker technologyused to detect the polymorphism. Marker types include but are notlimited to, e.g., detection of restriction fragment length polymorphisms(RFLP), detection of isozyme markers, randomly amplified polymorphic DNA(RAPD), amplified fragment length polymorphisms (AFLPs), detection ofsimple sequence repeats (SSRs), detection of amplified variablesequences of the plant genome, detection of self-sustained sequencereplication, or detection of single nucleotide polymorphisms (SNPs).SNPs can be detected e.g. via DNA sequencing, PCR-based sequencespecific amplification methods, detection of polynucleotidepolymorphisms by allele specific hybridization (ASH), dynamicallele-specific hybridization (DASH), molecular beacons, microarrayhybridization, oligonucleotide ligase assays, Flap endonucleases, 5′endonucleases, primer extension, single strand conformation polymorphism(SSCP) or temperature gradient gel electrophoresis (TGGE). DNAsequencing, such as the pyrosequencing technology has the advantage ofbeing able to detect a series of linked SNP alleles that constitute ahaplotype. Haplotypes tend to be more informative (detect a higher levelof polymorphism) than SNPs.

A “marker allele”, alternatively an “allele of a marker locus”, canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population.

“Marker assisted selection” (of MAS) is a process by which individualplants are selected based on marker genotypes.

“Marker assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

A “marker haplotype” refers to a combination of alleles at a markerlocus.

A “marker locus” is a specific chromosome location in the genome of aspecies where a specific marker can be found. A marker locus can be usedto track the presence of a second linked locus, e.g., one that affectsthe expression of a phenotypic trait. For example, a marker locus can beused to monitor segregation of alleles at a genetically or physicallylinked locus.

A “marker probe” is a nucleic acid sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence, through nucleic acidhybridization. Marker probes comprising 30 or more contiguousnucleotides of the marker locus (“all or a portion” of the marker locussequence) may be used for nucleic acid hybridization. Alternatively, insome aspects, a marker probe refers to a probe of any type that is ableto distinguish (i.e., genotype) the particular allele that is present ata marker locus.

The term “molecular marker” may be used to refer to a genetic marker, asdefined above, or an encoded product thereof (e.g., a protein) used as apoint of reference when identifying a linked locus. A marker can bederived from genomic nucleotide sequences or from expressed nucleotidesequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encodedpolypeptide. The term also refers to nucleic acid sequencescomplementary to or flanking the marker sequences, such as nucleic acidsused as probes or primer pairs capable of amplifying the markersequence. A “molecular marker probe” is a nucleic acid sequence ormolecule that can be used to identify the presence of a marker locus,e.g., a nucleic acid probe that is complementary to a marker locussequence. Alternatively, in some aspects, a marker probe refers to aprobe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Nucleic acids are“complementary” when they specifically hybridize in solution, e.g.,according to Watson-Crick base pairing rules. Some of the markersdescribed herein are also referred to as hybridization markers whenlocated on an indel region, such as the non-collinear region describedherein. This is because the insertion region is, by definition, apolymorphism vis a vis a plant without the insertion. Thus, the markerneed only indicate whether the indel region is present or absent. Anysuitable marker detection technology may be used to identify such ahybridization marker, e.g. SNP technology is used in the examplesprovided herein.

An allele “negatively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that a desired trait ortrait form will not occur in a plant comprising the allele.

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably and refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A “nucleotide” is amonomeric unit from which DNA or RNA polymers are constructed, andconsist of a purine or pyrimidine base, a pentose, and a phosphoric acidgroup. Nucleotides (usually found in their 5′-monophosphate form) arereferred to by their single letter designation as follows: “A” foradenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The term “phenotype”, “phenotypic trait”, or “trait” can refer to theobservable expression of a gene or series of genes. The phenotype can beobservable to the naked eye, or by any other means of evaluation knownin the art, e.g., weighing, counting, measuring (length, width, angles,etc.), microscopy, biochemical analysis, or an electromechanical assay.In some cases, a phenotype is directly controlled by a single gene orgenetic locus, i.e., a “single gene trait” or a “simply inheritedtrait”. In the absence of large levels of environmental variation,single gene traits can segregate in a population to give a “qualitative”or “discrete” distribution, i.e. the phenotype falls into discreteclasses. In other cases, a phenotype is the result of several genes andcan be considered a “multigenic trait” or a “complex trait”. Multigenictraits segregate in a population to give a “quantitative” or“continuous” distribution, i.e. the phenotype cannot be separated intodiscrete classes. Both single gene and multigenic traits can be affectedby the environment in which they are being expressed, but multigenictraits tend to have a larger environmental component.

A “physical map” of the genome is a map showing the linear order ofidentifiable landmarks (including genes, markers, etc.) on chromosomeDNA. However, in contrast to genetic maps, the distances betweenlandmarks are absolute (for example, measured in base pairs or isolatedand overlapping contiguous genetic fragments) and not based on geneticrecombination (that can vary in different populations).

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant.

A maize plant “derived from an inbred in the Stiff Stalk Syntheticpopulation” may be a hybrid.

A “polymorphism” is a variation in the DNA between two or moreindividuals within a population. A polymorphism preferably has afrequency of at least 1% in a population. A useful polymorphism caninclude a single nucleotide polymorphism (SNP), a simple sequence repeat(SSR), or an insertion/deletion polymorphism, also referred to herein asan “indel”.

An allele “positively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that the desired traitor trait form will occur in a plant comprising the allele.

The “probability value” or “p-value” is the statistical likelihood thatthe particular combination of a phenotype and the presence or absence ofa particular marker allele is random. Thus, the lower the probabilityscore, the greater the likelihood that a locus and a phenotype areassociated. The probability score can be affected by the proximity ofthe first locus (usually a marker locus) and the locus affecting thephenotype, plus the magnitude of the phenotypic effect (the change inphenotype caused by an allele substitution). In some aspects, theprobability score is considered “significant” or “nonsignificant”. Insome embodiments, a probability score of 0.05 (p=0.05, or a 5%probability) of random assortment is considered a significant indicationof association. However, an acceptable probability can be anyprobability of less than 50% (p=0.5). For example, a significantprobability can be less than 0.25, less than 0.20, less than 0.15, lessthan 0.1, less than 0.05, less than 0.01, or less than 0.001.

A “production marker” or “production SNP marker” is a marker that hasbeen developed for high-throughput purposes. Production SNP markers aredeveloped to detect specific polymorphisms and are designed for use witha variety of chemistries and platforms. The marker names used here beginwith a PHM prefix to denote ‘Pioneer Hi-Bred Marker’, followed by anumber that is specific to the sequence from which it was designed,followed by a “.” or a “−” and then a suffix that is specific to the DNApolymorphism. A marker version can also follow (A, B, C etc.) thatdenotes the version of the marker designed to that specificpolymorphism.

The term “progeny” refers to the offspring generated from a cross.

A “progeny plant” is a plant generated from a cross between two plants.

The term “quantitative trait locus” or “QTL” refers to a region of DNAthat is associated with the differential expression of a quantitativephenotypic trait in at least one genetic background, e.g., in at leastone breeding population. The region of the QTL encompasses or is closelylinked to the gene or genes that affect the trait in question. An“allele of a QTL” (or “QTL allele”) can comprise multiple genes or othergenetic factors within a contiguous genomic region or linkage group. Anallele of a QTL can be defined by a haplotype within a specified windowwherein said window is a contiguous genomic region that can be defined,and tracked, with a set of one or more polymorphic markers. Thehaplotype is then defined by the unique fingerprint of alleles at eachmarker within the specified window.

A “reference sequence” or a “consensus sequence” is a defined sequenceused as a basis for sequence comparison. The reference sequence for aPHM marker is obtained by sequencing a number of lines at the locus,aligning the nucleotide sequences in a sequence alignment program (e.g.Sequencher), and then obtaining the most common nucleotide sequence ofthe alignment. Polymorphisms found among the individual sequences areannotated within the consensus sequence. A reference sequence is notusually an exact copy of any individual DNA sequence, but represents anamalgam of available sequences and is useful for designing primers andprobes to polymorphisms within the sequence.

In “repulsion” phase linkage, the “favorable” allele at the locus ofinterest is physically linked with an “unfavorable” allele at theproximal marker locus, and the two “favorable” alleles are not inheritedtogether (i.e., the two loci are “out of phase” with each other).

“Northern leaf blight” (NLB), sometimes referred to as northern cornleaf blight (NCLB), is the disease caused by the pathogen Exserohilumturcicum. The disease, characterized by cigar-shaped lesions on leaftissue, can have severe effects on yield, particularly in tropicalclimates or during wet seasons in temperate climates.

As used herein, “northern leaf blight resistance” refers to enhancedresistance or tolerance to a fungal pathogen that causes northern leafblight when compared to a control plant. Effects may vary from a slightincrease in tolerance to the effects of the fungal pathogen (e.g.,partial inhibition) to total resistance such that the plant isunaffected by the presence of the fungal pathogen. An increased level ofresistance against a particular fungal pathogen or against a widerspectrum of fungal pathogens constitutes “enhanced” or improved fungalresistance. The embodiments of the disclosure will enhance or improveresistance to the fungal pathogen that causes northern leaf blight, suchthat the resistance of the plant to a fungal pathogen or pathogens willincrease. The term “enhance” refers to improve, increase, amplify,multiply, elevate, raise, and the like.

A “topeross test” is a test performed by crossing each individual (e.g.a selection, inbred line, clone or progeny individual) with the samepollen parent or “tester”, usually a homozygous line.

The phrase “under stringent conditions” refers to conditions under whicha probe or polynucleotide will hybridize to a specific nucleic acidsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.

Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is at least about 30° C.for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C.for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionsare often: 50% formamide, 5× SC, and 1% SDS, incubating at 42° C., or,5× SC, 1° A SDS, incubating at 65° C., with wash in 0.2× SSC, and 0.1° ASDS at 65° C. For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C., depending on primer length. Additionalguidelines for determining hybridization parameters are provided innumerous references.

An “unfavorable allele” of a marker is a marker allele that segregateswith the unfavorable plant phenotype, therefore providing the benefit ofidentifying plants that can be removed from a breeding program orplanting.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofmaize is commonly measured in bushels of seed per acre or metric tons ofseed per hectare per season. Yield is affected by both genetic andenvironmental factors. “Agronomics”, “agronomic traits”, and “agronomicperformance” refer to the traits (and underlying genetic elements) of agiven plant variety that contribute to yield over the course of growingseason. Individual agronomic traits include emergence vigor, vegetativevigor, stress tolerance, disease resistance or tolerance, herbicideresistance, branching, flowering, seed set, seed size, seed density,standability, threshability and the like. Yield is, therefore, the finalculmination of all agronomic traits.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the CLUSTAL V method of alignment(Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the CLUSTAL V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the CLUSTAL V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Genetic Mapping—Identification of Genetic Loci Associated with EnhancedResistance to Helminthosporium turcicum

It has been recognized for quite some time that specific genetic locicorrelating with particular phenotypes, such as resistance to northernleaf blight, can be mapped in an organism's genome. The plant breedercan advantageously use the genetic loci (i.e. molecular markers) toidentify desired individuals by detecting alleles at the loci that showa statistically significant probability of co-segregation with a desiredphenotype, manifested as linkage disequilibrium. By identifying amolecular marker or clusters of molecular markers that co-segregate witha trait of interest, a breeder is able to rapidly select a desiredphenotype by selecting for the proper molecular marker allele (a processcalled marker-assisted selection, or MAS). Such markers could also beused by breeders to design genotypes in silico and to practice wholegenome selection.

A variety of methods well known in the art are available for detectingmolecular markers or clusters of molecular markers that co-segregatewith a trait of interest, such as resistance to northern leaf blight.The basic idea underlying these methods is the detection of markers, forwhich alternative genotypes (or alleles) have significantly differentaverage phenotypes. Thus, one makes a comparison among marker loci ofthe magnitude of difference among alternative genotypes (or alleles) orthe level of significance of that difference. Trait genes are inferredto be located nearest the marker(s) that have the greatest associatedgenotypic difference.

Two such methods used to detect trait loci of interest are: 1)Population-based association analysis and 2) Traditional linkageanalysis. In a population-based association analysis, lines are obtainedfrom pre-existing populations with multiple founders, e.g. elitebreeding lines. Population-based association analyses rely on the decayof linkage disequilibrium (LD) and the idea that in an unstructuredpopulation, only correlations between genes controlling a trait ofinterest and markers closely linked to those genes will remain after somany generations of random mating. In reality, most pre-existingpopulations have population substructure. Thus, the use of a structuredassociation approach helps to control population structure by allocatingindividuals to populations using data obtained from markers randomlydistributed across the genome, thereby minimizing disequilibrium due topopulation structure within the individual populations (also calledsubpopulations). The phenotypic values are compared to the genotypes(alleles) at each marker locus for each line in the subpopulation. Asignificant marker-trait association indicates the close proximitybetween the marker locus and one or more genetic loci that are involvedin the expression of that trait.

The same principles underlie traditional linkage analysis; however, LDis generated by creating a population from a small number of founders.The founders are selected to maximize the level of polymorphism withinthe constructed population, and polymorphic sites are assessed for theirlevel of cosegregation with a given phenotype. A number of statisticalmethods have been used to identify significant marker-traitassociations. One such method is an interval mapping approach (Landerand Botstein, Genetics 121:185-199 (1989), in which each of manypositions along a genetic map (say at 1 cM intervals) is tested for thelikelihood that a gene controlling a trait of interest is located atthat position. The genotype/phenotype data are used to calculate foreach test position a LOD score (log of likelihood ratio). When the LODscore exceeds a threshold value, there is significant evidence for thelocation of a gene controlling the trait of interest at that position onthe genetic map (which will fall between two particular marker loci).

Molecular marker loci that demonstrate statistically significantco-segregation with resistance to northern leaf blight, as determined byassociation mapping and traditional linkage mapping techniques, areprovided herein. Detection of these marker loci or additional linkedmarker loci can be used in marker-assisted maize breeding programs toproduce plants with enhanced resistance to northern leaf blight or toeliminate plants that do not have enhanced resistance to northern leafblight from breeding programs or planting.

Markers Associated with Resistance to Northern Leaf Blight

Methods involving detecting the presence of one or more marker alleles(at one or more marker loci) associated with enhanced resistance tonorthern leaf blight in the germplasm of the maize plant are providedherein. The maize plant can be a hybrid or inbred.

The marker locus can be selected from any of the marker loci providedherein including but not limited to: PHM16750, PHM15741, PHM16854,PHM3870, PHM14018, PHM18056, PHM3467, PHM7958, PZE-105068275,PZE-105068432, PZE-105068572, SYN30642, PZE-105068746, PZE-105069095,PZE-105069706, PZE-105069906, and PZE-105070525; as well as any othermarker linked to these markers (linked markers can be determined fromthe MaizeGDB resource).

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or in centiMorgans (cM). The cM is a unit ofmeasure of genetic recombination frequency. One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a traitat another locus due to crossing over in a single generation (meaningthe traits segregate together 99% of the time). Because chromosomaldistance is approximately proportional to the frequency of crossing overevents between traits, there is an approximate physical distance thatcorrelates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, one cM is equal to a 1% chance that a marker locuswill be separated from another locus, due to crossing over in a singlegeneration.

The closer a marker is to a gene controlling a trait of interest, themore effective and advantageous that marker is as an indicator for thedesired trait. Closely linked loci display an inter-locus cross-overfrequency of about 10% or less, preferably about 9% or less, still morepreferably about 8% or less, yet more preferably about 7% or less, stillmore preferably about 6% or less, yet more preferably about 5% or less,still more preferably about 4% or less, yet more preferably about 3% orless, and still more preferably about 2% or less. In highly preferredembodiments, the relevant loci (e.g., a marker locus and a target locus)display a recombination frequency of about 1% or less, e.g., about 0.75%or less, more preferably about 0.5% or less, or yet more preferablyabout 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or lessapart. Put another way, two loci that are localized to the samechromosome, and at such a distance that recombination between the twoloci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be“proximal to” each other.

Although particular marker alleles can show co-segregation with thenorthern leaf blight resistance phenotype, it is important to note thatthe marker locus is not necessarily responsible for the expression ofthe northern leaf blight resistance phenotype. For example, it is not arequirement that the marker polynucleotide sequence be part of a genethat imparts enhanced northern leaf blight resistance (for example, bepart of the gene open reading frame). The association between a specificmarker allele and the enhanced northern leaf blight resistance phenotypeis due to the original “coupling” linkage phase between the markerallele and the allele in the ancestral maize line from which the alleleoriginated. Eventually, with repeated recombination, crossing overevents between the marker and genetic locus can change this orientation.For this reason, the favorable marker allele may change depending on thelinkage phase that exists within the resistant parent used to createsegregating populations. This does not change the fact that the markercan be used to monitor segregation of the phenotype. It only changeswhich marker allele is considered favorable in a given segregatingpopulation.

Chromosomal Intervals

Chromosomal intervals that correlate with northern leaf blightresistance are provided. A variety of methods well known in the art areavailable for identifying chromosomal intervals. The boundaries of suchchromosomal intervals are drawn to encompass markers that will be linkedto the gene controlling the trait of interest. In other words, thechromosomal interval is drawn such that any marker that lies within thatinterval (including the terminal markers that define the boundaries ofthe interval) can be used as a marker for northern leaf blightresistance. Each interval comprises at least one QTL, and furthermore,may indeed comprise more than one QTL. Close proximity of multiple QTLin the same interval may obfuscate the correlation of a particularmarker with a particular QTL, as one marker may demonstrate linkage tomore than one QTL. Conversely, e.g., if two markers in close proximityshow co-segregation with the desired phenotypic trait, it is sometimesunclear if each of those markers identifies the same QTL or twodifferent QTL. Regardless, knowledge of how many QTL are in a particularinterval is not necessary to make or practice the subject materialpresented herein.

An interval on chromosome 5 containing one or more QTL associated withnorthern leaf blight resistance may be defined by and includes: PHM18056and PHM7958. The interval may further be refined to a chromosomalinterval defined by and including PZE-105068275 and PZE-105070525, whichrepresents a subinterval of the chromosomal interval defined by andincluding PHM18056 and PHM7958. Any marker located within any of theseintervals finds use as a marker for northern leaf blight resistance inmaize.

Chromosomal intervals can also be defined by markers that are linked to(show linkage disequilibrium with) a marker of interest, and r² is acommon measure of linkage disequilibrium (LD) in the context ofassociation studies. If the r² value of LD between any marker locusidentified herein and another marker within the chromosome 5 interval(also described herein) is greater than ⅓ (Ardlie et al., Nature ReviewsGenetics 3:299-309 (2002)), the loci are linked.

Marker Alleles and Haplotype Combinations

A haplotype, or a combination of alleles at one or more marker loci, canrepresent the genetic signature of a QTL allele. Any of the markeralleles described herein could be used alone or in combination toidentify and select maize plants with enhanced northern leaf blight byidentifying a haplotype representative of a QTL allele as could anymarker allele in linkage disequilibrium with the marker allelesdescribed herein. The marker alleles representative of the QTL allelemay include: a “G” at PZE-105068275; an “A” at PZE-105068432; a “C” atPZE-105068572; a “T” at SYN30642; a “C” at PZE-105068746; an “A” atPZE-105069095; an “A” at PZE-105069706; a “T” at PZE-105069906; and/or a“C” at PZE-105070525.

Methods for identifying maize plants with northern leaf blightresistance are provided herein. The methods involve analyzing DNA of amaize plant for the presence of a QTL allele associated with northernleaf blight resistance and selecting maize plants as having northernleaf blight resistance if the QTL allele is detected.

The QTL allele may comprise any of the following marker alleles alone orin combination: a “G” at PZE-105068275; an “A” at PZE-105068432; a “C”at PZE-105068572; a “T” at SYN30642; a “C” at PZE-105068746; an “A” atPZE-105069095; an “A” at PZE-105069706; a “T” at PZE-105069906; and a“C” at PZE-105070525.

In one aspect, the QTL allele is located on chromosome 5 in achromosomal interval defined by and including PHM18056 and PHM7958. Inanother aspect, the QTL allele is located on chromosome 5 in achromosomal interval defined by and including PZE-105068275 andPZE-105070525, which is a subinterval of the PHM18056 and PHM7958interval.

The skilled artisan would expect that there are additional polymorphicsites at marker loci in and around the chromosome 5 markers identifiedherein, wherein one or more polymorphic sites is in linkagedisequilibrium (LD) with one or more of the polymorphic sites in therepresentative haplotype. Two particular alleles at differentpolymorphic sites are said to be in LD if the presence of the allele atone of the sites tends to predict the presence of the allele at theother site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).Detecting the presence of a QTL allele does not in any way mean that theQTL allele can only be defined by the haplotype comprising: a “G” atPZE-105068275; an “A” at PZE-105068432; a “C” at PZE-105068572; a “T” atSYN30642; a “C” at PZE-105068746; an “A” at PZE-105069095; an “A” atPZE-105069706; a “T” at PZE-105069906; and a “C” at PZE-105070525.Rather, the presence of the QTL allele can be detected using any of themarker alleles defined herein alone or in combination and/or any othermarker allele within the specified chromosomal interval that is inlinkage disequilibrium with any of the marker alleles defined herein.

Marker-Assisted Selection (MAS)

Molecular markers can be used in a variety of plant breedingapplications (e.g. see Staub et al. (1996) Hortscience 31: 729-741;Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of themain areas of interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection (MAS). A molecularmarker that demonstrates linkage with a locus affecting a desiredphenotypic trait provides a useful tool for the selection of the traitin a plant population. This is particularly true where the phenotype ishard to assay, e.g. many disease resistance traits, or, occurs at a latestage in plant development, e.g. kernel characteristics. Since DNAmarker assays are less laborious and take up less physical space thanfield phenotyping, much larger populations can be assayed, increasingthe chances of finding a recombinant with the target segment from thedonor line moved to the recipient line. The closer the linkage, the moreuseful the marker, as recombination is less likely to occur between themarker and the gene causing the trait, which can result in falsepositives. Having flanking markers decreases the chances that falsepositive selection will occur as a double recombination event would beneeded. The ideal situation is to have a marker in the gene itself, sothat recombination cannot occur between the marker and the gene. Such amarker is called a ‘perfect marker’.

When a gene is introgressed by MAS, it is not only the gene that isintroduced but also the flanking regions (Gepts. (2002). Crop Sci; 42:1780-1790). This is referred to as “linkage drag.” In the case where thedonor plant is highly unrelated to the recipient plant, these flankingregions carry additional genes that may code for agronomicallyundesirable traits. This “linkage drag” may also result in reduced yieldor other negative agronomic characteristics even after multiple cyclesof backcrossing into the elite maize line. This is also sometimesreferred to as “yield drag.” The size of the flanking region can bedecreased by additional backcrossing, although this is not alwayssuccessful, as breeders do not have control over the size of the regionor the recombination breakpoints (Young et al. (1998) Genetics120:579-585). In classical breeding it is usually only by chance thatrecombinations are selected that contribute to a reduction in the sizeof the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264).Even after 20 backcrosses in backcrosses of this type, one may expect tofind a sizeable piece of the donor chromosome still linked to the genebeing selected. With markers however, it is possible to select thoserare individuals that have experienced recombination near the gene ofinterest. In 150 backcross plants, there is a 95% chance that at leastone plant will have experienced a crossover within 1 cM of the gene,based on a single meiosis map distance. Markers will allow unequivocalidentification of those individuals. With one additional backcross of300 plants, there would be a 95% chance of a crossover within 1 cMsingle meiosis map distance of the other side of the gene, generating asegment around the target gene of less than 2 cM based on a singlemeiosis map distance. This can be accomplished in two generations withmarkers, while it would have required on average 100 generations withoutmarkers (See Tanksley et al., supra). When the exact location of a geneis known, flanking markers surrounding the gene can be utilized toselect for recombinations in different population sizes. For example, insmaller population sizes, recombinations may be expected further awayfrom the gene, so more distal flanking markers would be required todetect the recombination.

The availability of integrated linkage maps of the maize genomecontaining increasing densities of public maize markers has facilitatedmaize genetic mapping and MAS. See, e.g. the IBM2 Neighbors maps, whichare available online on the MaizeGDB website.

The key components to the implementation of MAS are: (i) Defining thepopulation within which the marker-trait association will be determined,which can be a segregating population, or a random or structuredpopulation; (ii) monitoring the segregation or association ofpolymorphic markers relative to the trait, and determining linkage orassociation using statistical methods; (iii) defining a set of desirablemarkers based on the results of the statistical analysis, and (iv) theuse and/or extrapolation of this information to the current set ofbreeding germplasm to enable marker-based selection decisions to bemade. The markers described in this disclosure, as well as other markertypes such as SSRs and FLPs, can be used in marker-assisted selectionprotocols.

In general, MAS for the purposes described herein uses polymorphicmarkers that have been identified as having a significant likelihood ofco-segregation with northern leaf blight resistance. Such markers arepresumed to map near a gene or genes that give the plant its northernleaf blight resistance phenotype, and are considered indicators for thedesired trait, or markers. Plants are tested for the presence of adesired allele in the marker, and plants containing a desired genotypeat one or more loci are expected to transfer the desired genotype, alongwith a desired phenotype, to their progeny.

Markers were identified from both linkage mapping and associationanalysis as being associated with resistance to northern leaf blight.Reference sequences for each of the markers are represented by SEQ IDNOs:1-17. The SNPs could be used alone or in combination (i.e. a SNPhaplotype) to select for a favorable QTL allele associated withresistance to northern leaf blight.

Methods for introgressing a QTL allele associated with northern leafblight resistance into a maize plant are provided herein. The methodsinvolve screening a population with at least one marker to determine ifone or more maize plants from the population comprises a QTL alleleassociated with northern leaf blight resistance and selecting from thepopulation one or more maize plants that have the QTL allele. The QTLallele may comprise: a “G” at PZE-105068275; an “A” at PZE-105068432; a“C” at PZE-105068572; a “T” at SYN30642; a “C” at PZE-105068746; an “A”at PZE-105069095; an “A” at PZE-105069706; a “T” at PZE-105069906; and a“C” at PZE-105070525.

The skilled artisan would expect that there might be additionalpolymorphic sites at marker loci in and around the chromosome 5 markersidentified herein, wherein one or more polymorphic sites is in linkagedisequilibrium (LD) with an allele at one or more of the polymorphicsites in the haplotype and thus could be used in a marker assistedselection program to introgress a QTL allele of interest. Two particularalleles at different polymorphic sites are said to be in LD if thepresence of the allele at one of the sites tends to predict the presenceof the allele at the other site on the same chromosome (Stevens, Mol.Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2cM, or 1 cM (on a single meiosis based genetic map) of the resistance tonorthern leaf blight QTL.

The skilled artisan would also understand that allelic frequency (andhence, haplotype frequency) can differ from one germplasm pool toanother. Germplasm pools vary due to maturity differences, heteroticgroupings, geographical distribution, etc. As a result, SNPs and otherpolymorphisms may not be informative in some germplasm pools.

Plant Compositions

Maize plants identified, selected, and/or generated by any of themethods described above are also of interest.

Seed Treatments

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the methods and compositions described herein. Seed materialcan be treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematicides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil,imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide,mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, PCNB,penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin,prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin,sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb,thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seeds that produce plants with specific traits (such as resistance tonorthern leaf blight) may be tested to determine which seed treatmentoptions and application rates may complement such plants in order toenhance yield. Further, the good root establishment and early emergencethat results from the proper use of a seed treatment may result in moreefficient nitrogen use, a better ability to withstand resistance tonorthern leaf blight and an overall increase in yield potential of aplant or plants containing a certain trait when combined with a seedtreatment.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed subject matter. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatpersons skilled in the art will recognize various reagents or parametersthat can be altered without departing from the scope of the appendedclaims.

Example 1 Phenotyping of Northern Leaf Blight Infection

Maize plants can be evaluated for Northern Leaf Blight (NLB) on a 1(highly susceptible) to 9 (highly resistant) scale, where scores of 1-3indicate “susceptible”, scores of 4-6 indicate “intermediate”, andscores of 7-9 indicate “resistant”. The scoring diagram in FIG. 1 can beused as a guide, with an emphasis placed on lesions above the ear. Thelesions can be verified as being caused by northern leaf blightinfection by checking that the lesions are cigar or boat-shaped withsmooth sides and/or by sending a sample to a diagnostic lab to confirmthe identity of the pathogen.

At two to four weeks after flowering, scores can be obtained from a fewknown susceptible lines and then compared to their historical scores. Ifthe known susceptible lines rate at least two scores higher than theirhistorical scores, scoring of the lines in the test set can be delayed,thereby allowing the disease to advance to a standard state ofinfection. The scoring period can only be extended until prior to plantsenescence. Thus, if the scores are still too high after 4-5 weeks, thedisease pressure is insufficient for effective scoring.

If scores from the known susceptible lines do correlate with theirhistorical scores in the time period from 2-4 weeks after floweringuntil prior to plant senescence, the test lines can be scored on a plotbasis using the scoring diagrams in FIG. 1 as a guide.

Example 2 Association Mapping Analysis

An association mapping strategy was undertaken to identify maize geneticmarkers associated with resistance to Northern Leaf Blight.

A collection of maize lines was analyzed using ILLUMINA® SNP Genotyping(a 1536-plex assay). SNP variation was used to generate specifichaplotypes across inbreds for regions of the genome. This data was usedfor identifying associations between alleles and northern leaf blightresistance at the genome level.

Resistance scores and genotypic information were incorporated into anassociation mapping analysis. A structure-based association analysis wasconducted using standard association mapping methods where thepopulation structure is controlled using marker data. Two chromosome 5markers, PHM16750 and PHM15741, were significantly associated with thenorthern leaf blight resistance trait in a non-Stiff Stalksubpopulation. In addition, three chromosome 5 markers, PHM16854,PHM3870, and PHM14018, were significantly associated with the northernleaf blight resistance trait in a tropical subpopulation. Table 1provides marker information for the chromosome 5 markers thatdemonstrated linkage disequilibrium with the northern leaf blightphenotype using the structured association mapping method.

TABLE 1 Maize markers significantly associated with resistance tonorthern leaf blight infection in structured association analysis Singlemeiosis IBM2 based genetic genetic map map Marker Reference positionposition Name sequence Subpop P-value (cM) (cM) PHM16750 SEQ ID NSS5.60E−04 96.3 N/A NO: 1 PHM15741 SEQ ID NSS 6.60E−04 96.7 289.3 NO: 2PHM16854 SEQ ID Tropical 0.0038 86.8 257.8 NO: 3 PHM3870 SEQ ID Tropical0.0046 91.7 271.5 NO: 4 PHM14018 SEQ ID Tropical 0.0047 96.1 289.3 NO: 5

In addition, an association analysis was performed on a set of Argentineinbreds. This association analysis also identified significant markertrait associations for subpopulation 2 in the same interval ofchromosome 5 (Table 2).

TABLE 2 Maize markers significantly associated with resistance tonorthern leaf blight infection in an association analysis performed on aset of Argentine inbreds Single meiosis based IBM2 genetic MarkerReference genetic map map Name sequence P-value position (cM) position(cM) PHM18056 SEQ ID 9.80E−05 87.1 N/A NO: 6 PHM3467 SEQ ID 2.26E−04 90N/A NO: 7 PHM7958 SEQ ID 5.20E−05 105.2 307 NO: 8

The statistical probabilities that the marker allele and phenotype aresegregating independently are reflected in the association mappingadjusted probability values in Tables 1 and 2, which is a probability(P) derived from analysis of association between genotype and phenotype.The lower the probability value, the more significant is the associationbetween the marker genotype at that locus and the northern leaf blightinfection tolerance phenotype.

Note. The results shown in Tables 1 and 2 are based on two independentsets of data. Table 2 shows the results from a single set of data fromArgentine inbreds.

Example 3 QTL Mapping Using Double Haploid Breeding Populations

A QTL interval mapping analysis was undertaken to identify chromosomeintervals and markers associated with northern leaf blight resistanceusing a population of 186 doubled haploids generated by a cross betweenPHBNB and PHFHH. Line PHBNB has greater resistance to northern leafblight infection than line PHFHH. The doubled haploid lines generatedfrom the cross were phenotyped under natural northern leaf blightinfection in a single growing season and in two locations. Maize doubledhaploid progeny were genotyped using a set of 768 SNPs distributed inthe maize genome.

A significant peak was identified on chromosome 5, between 90 to 100 cMon the internally derived single meiosis based genetic map, indicatingthat the region houses one or more QTL associated with resistance tonorthern leaf blight. Using interval mapping analysis, a number ofmarkers showed association with the phenotype at a confidence level of p<0.05. This finding concurred with the other Examples, showing that thedifferent approaches identify the same region. Table 3 shows the geneticeffects for the QTL on chromosome 5, position 90-100 cM.

TABLE 3 Haplotypic effects for PHBNB × PHFHH cross Haplotype Average ofChr5 90-100 cM NLFBLT n Favorable 5.78 138 (from PHBNB) Unfavorable 4.9886 (from PHFHH)

EXAMPLE 4 High-Resolution Gene Mapping and Near Isogenic Lines andHybrids

High-resolution gene mapping by progeny testing of homozygousrecombinant plants was undertaken to further refine the northern leafblight resistance QTL. A mapping population was created from the crossof PH890 RC1 and inbred PHBNB. Another population for fine mapping wascreated from the cross of inbreds PHFHH and PHBD6. The PH890 RC1×PHBNBpopulation consisted of 94 BC₅F₃ families generated by selfing andfixing selected recombinant BC₅ plants from a total of approximately3000 BC₅ plants harbouring a heterozygous fragment at the region from 90to 105 cM on chromosome 5. This strategy permitted coverage withrecombinants of the whole QTL region. The PHFHH×PHBD6 populationconsisted of 37 BC₄F₃ families generated by selfing and fixing selectedrecombinant BC₄ plants from a total of approximately 3000 BC₄ plantsharbouring a heterozygous fragment at the region from 90 to 105 cM onchromosome 5.

BC₅F₃ and BC₄F₃ near-isogenic lines (NIL) harbouring allelic variationat the region of the preferred markers were generated by marker assistedselection for both crosses. The NILs were generated by introgressing theQTL region from PHBNB or PHBD6 into recurrent parents, cleaning thegenetic background, and selecting specific recombinants at the region ofthe preferred markers. By selfing individual BC₄F₂/BC₅F₂ plantsharbouring a heterozygous fragment at the region of the preferredmarkers, negative and positive near-isogenic lines were derived, and theQTL was treated as a single Mendelian factor.

Phenotypic Scoring

Phenotypic scoring of each of the different families from the crossinvolving PH890 RC1×PHBNB and from PHFHH×PHBD6 cross, the parents of thecrosses and the generated NILs, was based on sets of phenotypic datacollected from the field (field experiments under natural infection;three locations) obtained in one crop season.

Maize Genotyping

Maize BC₅F₃ progeny from the PH890 RC1×PHBNB cross and BC₄F₃ progenyfrom PHFHH×PHBD6, the parents of the crosses, and the generated NILswere genotyped using polymorphic SNPs at the QTL region on chromosome 5.

Windows QTL Cartographer was used for both the marker regressionanalysis and QTL interval mapping. LOD scores (logarithm of the oddsratio) were estimated across the target regions according the standardQTL mapping procedures.

Mean scores were used in QTL interval mapping. The LOD threshold was2.5. A confidence interval was estimated for each QTL. As thesepopulations were generated by marker assisted selection (not randomevents of recombination), marker regression analysis was considered aspowerful as interval mapping analysis.

Near Isogenic Lines and Fine Mapping

The near isogenic genetic materials harbouring allelic variation at theregion of preferred markers (93.3-96.8 cM on the proprietary singlemeiosis based genetic map) showed a significant difference in theirresponse to the disease in both Argentina and USA. Additional markerloci were evaluated in an increased number of individuals in an effortto further narrow the QTL region. The region housing the QTL was furtherrefined to a region between and including markers PZE-105068275(reference sequence is represented by SEQ ID NO:9) and PZE-105070525(reference sequence is represented by SEQ ID NO:17), which are locatedat 96.7 and 97.5 cM, respectively.

SNP Haplotype

Table 4 shows the genotype of SNPs at the region of preferred markersfor the favorable resistant haplotype.

TABLE 4 SNP Haplotypes Genetic Map Ref SNP Marker Position SNP SeqPosition PZE-105068275 96.67 G SEQ 51 ID NO: 9 PZE-105068432 96.72 A SEQ51 ID NO: 10 PZE-105068572 96.78 C SEQ 51 ID NO: 11 SYN30642 96.79 T SEQ61 ID NO: 12 PZE-105068746 96.84 C SEQ 51 ID NO: 13 PZE-105069095 97.02A SEQ 51 ID NO: 14 PZE-105069706 97.31 A SEQ 51 ID NO: 15 PZE-10506990697.39 T SEQ 51 ID NO: 16 PZE-105070525 97.45 C SEQ 51 ID NO: 17

This present study has identified chromosome intervals and individualmarkers that correlate with northern leaf blight resistance. Markersthat lie within these intervals are useful for use in MAS, as well asother purposes.

What is claimed:
 1. A method of identifying a maize plant with northernleaf blight resistance comprising: a. analyzing DNA of a maize plant forthe presence of a QTL allele associated with northern leaf blightresistance, wherein said QTL allele is located within an interval onchromosome 5 comprising and flanked by PHM18056 and PHM7958 and said QTLallele comprises: i. a “G” at PZE-105068275; ii. an “A” atPZE-105068432; iii. a “C” at PZE-105068572; iv. a “T” at SYN30642; v. a“C” at PZE-105068746; vi. an “A” at PZE-105069095; vii. an “A” atPZE-105069706; viii. a “T” at PZE-105069906; and ix. a “C” atPZE-105070525; b. selecting said maize plant if said QTL allele isdetected.
 2. The method of claim 1, wherein said QTL allele is locatedwithin an interval on chromosome 5 defined by and includingPZE-105068275 and PZE-105070525.
 3. A method of introgressing a QTLallele associated with northern leaf blight resistance into a maizeplant said method comprising: a. screening a population with at leastone marker to determine if one or more maize plants from the populationcomprises a QTL allele associated with northern leaf blight resistance,wherein the QTL allele comprises: i. a “G” at PZE-105068275; ii. an “A”at PZE-105068432; iii. a “C” at PZE-105068572; iv. a “T” at SYN30642; v.a “C” at PZE-105068746; vi. an “A” at PZE-105069095; vii. an “A” atPZE-105069706; viii. a “T” at PZE-105069906; and ix. a “C” atPZE-105070525; and b. selecting from said population a maize plantcomprising the QTL allele.